EP0149162A2 - Variety of Bacillus thuringiensis effective against Coleoptera and an insecticidally active preparation obtainable therefrom, or a toxine, and their use to combate Coleoptera - Google Patents
Variety of Bacillus thuringiensis effective against Coleoptera and an insecticidally active preparation obtainable therefrom, or a toxine, and their use to combate Coleoptera Download PDFInfo
- Publication number
- EP0149162A2 EP0149162A2 EP84115535A EP84115535A EP0149162A2 EP 0149162 A2 EP0149162 A2 EP 0149162A2 EP 84115535 A EP84115535 A EP 84115535A EP 84115535 A EP84115535 A EP 84115535A EP 0149162 A2 EP0149162 A2 EP 0149162A2
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- EP
- European Patent Office
- Prior art keywords
- bacillus thuringiensis
- coleoptera
- toxin
- pathotype
- obtainable therefrom
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 241000193388 Bacillus thuringiensis Species 0.000 title claims abstract description 19
- 229940097012 bacillus thuringiensis Drugs 0.000 title claims abstract description 19
- 241000254173 Coleoptera Species 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title description 3
- 239000003053 toxin Substances 0.000 claims abstract description 20
- 231100000765 toxin Toxicity 0.000 claims abstract description 20
- 239000002917 insecticide Substances 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 4
- 239000013078 crystal Substances 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 230000009089 cytolysis Effects 0.000 claims description 2
- 241000193369 Bacillus thuringiensis serovar tenebrionis Species 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 3
- 108700012359 toxins Proteins 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 7
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- 230000000749 insecticidal effect Effects 0.000 description 6
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- 230000000694 effects Effects 0.000 description 4
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- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000256837 Apidae Species 0.000 description 1
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- 108010053835 Catalase Proteins 0.000 description 1
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- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
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- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108010048581 Lysine decarboxylase Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108090000913 Nitrate Reductases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 241000382353 Pupa Species 0.000 description 1
- 241001340903 Pyralis farinalis Species 0.000 description 1
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 1
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- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 1
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- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
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- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
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- 230000037029 cross reaction Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 210000005027 intestinal barrier Anatomy 0.000 description 1
- 230000007358 intestinal barrier function Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 1
- 229940120668 salicin Drugs 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000004563 wettable powder Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/075—Bacillus thuringiensis
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/832—Bacillus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/82—Proteins from microorganisms
- Y10S530/825—Bacteria
Definitions
- Bacillus thuringiensis was first described in 1915 as a cause of the flaccid addiction of flour moth larvae. Until the 1970s, all isolates from Bt belonged to a pathotype that was only effective against larvae of Lepidoptera. This pathotype is called type A. In addition to the var. Thuringiensis, it also includes the varieties kurstaki and galleriae, which are also used in practice.
- bacterial insecticides act very selectively on certain types of pests.
- US Pat. No. 4,166,112 claims an insecticide that contains the variety Bacillus thuringiensis var. Israelensis found by Goldberg as an active ingredient. This shows very clearly how selectively this bacterial strain is pathogenic against mosquito larvae, while other organisms are not affected.
- the invention relates to a new variety of Bacillus thuringiensis, which is referred to as Bacillus thuringiensis var. Tenebrionis (Btt). It could be shown that this strain has an insecticidal action, namely on beetles (Coleoptera), in particular against chrysomelids, such as. B. has the blue alder beetle (Agelastica alni) and the Colorado beetle (Leptinotarsa decemlineata). It is a new pathotype, which is called pathotype C.
- Another object of the invention is a bacterial insecticide which contains as active ingredient Bacillus thuringiensis of pathotype C, in particular Bacillus thuringiensis var. Tenebrionis, or an insecticidal preparation or toxin obtainable therefrom.
- the new variety Btt was isolated from the developmental stages of the common flour beetle (Tenebrio molitor L., Coleoptera: Tenebrionidae). The pure culture is kept at 25-30 o C on nutrient agar in slant tubes. Dry spores were frozen for type preservation. The determination was based on morphological and biochemical characteristics.
- the isolated bacterium produces large, rough, whitish colonies on nutrient agar, the cells of which sporulate after 18 to 24 hours.
- the vegetative cell is flagellate, rod-shaped and measures approximately 1.0 x 4 - 8 ⁇ m. She reacts gram positive.
- the spore mother cell contains a parasporal crystal of flat, plate-like shape and with the outline of a parallelogram, rhombus or square (approx. 0.8 - 1, 5 ⁇ m edge length).
- Bacillus growth is optionally anaerobic. In contrast, sporulation is strictly aerobic.
- the optimum temperature for the growth of vegetative cells is 25-30 ° C.
- Autoclaving for 15 minutes at 121 ° C. (corresponding to 1 bar overpressure) completely inactivates the spores and also the crystal toxin.
- UV radiation at 254 nm inactivates the spores at the appropriate dose, but not the crystal toxin.
- the biochemical performances of the vegetative cells are as follows.
- Degradable carbohydrates such as glucose
- mannose and sucrose are broken down under acidification, but not lactose, cellobiose, salicin, arabinose, xylose or mannitol.
- Aesculin is hydrolyzed, as is starch.
- the following enzymes can be detected: catalase, nitrate reductase and proteinase. The following could not be detected: lysine decarboxylase, phenylalanine deaminase, urease and lecithinase.
- a brooder was used for production purposes on a laboratory scale. 50 ml of medium were fermented in 250 ml Erlenmeyer flasks. The medium had the following composition: yeast extract, tryptone 0.5%, glucose 0, 1%, KH 2 P0 4 0.08% in tap water 0.5% p H 7.0. 25-30 ° C was selected as the incubation temperature. Following the sporulation of the culture, which can be checked microscopically, the biomass was centrifuged off and washed out with water or buffer. The effect of Btt on coleopteran larvae follows the scheme already known from pathotype A: feeding stops within a few hours and mortality occurs after a few days depending on the dose.
- the toxic effect is due to the fact that the epithelium of the middle intestine is permanently damaged by the crystal toxin dissolved in the intestinal juice.
- the damaged intestinal cells detach from one another and from the basement membrane and drift into the intestinal lumen.
- bacterial cells can penetrate the hemocoel and there complete the process of disintegration through fatal septicemia.
- the new variety Btt, pathotype C whose prototype could be isolated from a dead flour beetle pupa, was deposited with the German Collection for Microorganisms under the number DSM 2803. After the serological examination, the var. Tenebrionis, strain DSM 2803, belongs to the serotype H 8a, 8b.
- the new variety Btt, pathotype C shows a selective insecticidal effect on Coleoptera, for example on the blue alder beetle or the Colorado beetle. The effect is much stronger for larvae than for adults. No effect was observed against Lepidoptera, for example against the meal moth (Ephestia Kühniella Zell.), Cabbage moth (Plutella xylostella L.) and also not against Diptera, for example the yellow fever mosquito (Aedes aegypti L.). The results of the bioassays carried out are summarized in the following table.
- the actual insecticidal principle of action in Bacillus thuringiensis is crystal toxin.
- the crystal mass can be isolated by separating and cleaning the crystals from the cell residues and the spores in the sporulated cultures after lysis of the sporangia, for example using density gradients.
- antibodies can be obtained which show no cross-reaction with other toxins of the pathotype A or B in the immunodiffusion test according to Ouchterlony.
- the toxin is encoded by a gene contained in a plasmid.
- the toxin is also produced in asporogenic mutants, which are generated by treatment with ethyl methane sulfonate.
- the ability to produce toxins can be transferred to other Bacillus thuringiensis varieties or other bacterial systems by plasmid or gene transfer.
- the insecticidal effect is based on the presence of the plasmid-encoded toxin.
- Bacillus thuringiensis var. Tenebrionis or an insecticidal preparation or toxin obtainable therefrom is mixed with conventional additives (carriers, adhesives, wetting agents, etc.) in a manner known per se and converted into a suitable application form.
- the insecticide formulated in this way can be used in the form of a wettable powder, a suspension, as granules or the like.
- a purified, sporulated culture is preferably used in the form of a suspension with the addition of a wetting agent, for example 0.1% Citowet R (BASF). 10 to 10 15 , preferably 10 13 to 10 14 spores and an equivalent number of toxic crystals are sprayed per hectare to be treated.
- a potato field can be protected from the Colorado beetle for 2 to 4 weeks.
- further treatments are required at the intervals mentioned.
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Tropical Medicine & Parasitology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
Neben den chemischen Insektiziden sind seit mehreren Jahren bakterielle Insektizide Gegenstand intensiver Forschungsarbeiten. Erstmals wurde 1915 der Bacillus thuringiensis (Bt) als Erreger der Schlaffsucht von Mehlmottenlarven beschrieben. Bis in die 70er Jahre gehörten alle Isolate von Bt einem Pathotyp an, der nur gegenüber Larven von Lepidopteren wirksam war. Dieser Pathotyp wird als Typ A bezeichnet. Zu ihm gehören außer der var. thuringiensis die ebenfalls praktisch benutzten Varietäten kurstaki und galleriae.In addition to chemical insecticides, bacterial insecticides have been the subject of intensive research for several years. Bacillus thuringiensis (Bt) was first described in 1915 as a cause of the flaccid addiction of flour moth larvae. Until the 1970s, all isolates from Bt belonged to a pathotype that was only effective against larvae of Lepidoptera. This pathotype is called type A. In addition to the var. Thuringiensis, it also includes the varieties kurstaki and galleriae, which are also used in practice.
1977 isolierten Goldberg und Margalit aus einer von Mückenbrutplätzen in der Negev-Wüste stammenden Probe einen Stamm, der sich gegenüber Larven verschiedener Nematoceren (Diptera) als pathogen erwies und der später als Bacillus thuringiensis var. israelensis (Bti) klassifiziert wurde. Dieser Pathotyp wird als Typ B bezeichnet. Auch diese Varietät wird inzwischen wirtschaftlich genutzt.In 1977 Goldberg and Margalit isolated from a sample from mosquito hatcheries in the Negev desert a strain that was found to be pathogenic to larvae of various nematocerae (Diptera) and was later classified as Bacillus thuringiensis var. Israelensis (Bti). This pathotype is called Type B. This variety is now also used economically.
Der Vorteil der bakteriellen Insektizide liegt darin, daß sie sehr selektiv auf bestimmte Schädlingsarten wirken.The advantage of bacterial insecticides is that they act very selectively on certain types of pests.
So wird beispielsweise in dem US-Patent 4.166.112 ein Insektizid beansprucht, das die von Goldberg gefundene Varietät Bacillus thuringiensis var. israelensis als aktiven Bestandteil enthält. Hierin wird sehr deutlich gezeigt, wie selektiv dieser Bakterienstamm gegen Mückenlarven pathogen wirkt, während andere Organismen nicht beeinflußt werden.For example, US Pat. No. 4,166,112 claims an insecticide that contains the variety Bacillus thuringiensis var. Israelensis found by Goldberg as an active ingredient. This shows very clearly how selectively this bacterial strain is pathogenic against mosquito larvae, while other organisms are not affected.
Es besteht weiterhin Bedarf an bakteriellen Insektiziden gegen andere Schädlingsgruppen.There is still a need for bacterial insecticides against other groups of pests.
Gegenstand der Erfindung ist eine neue Varietät des Bacillus thuringiensis, die als Bacillus thuringiensis var. tenebrionis (Btt) bezeichnet wird. Es konnte gezeigt werden, daß dieser Stamm eine insektizide Wirkung, und zwar auf Käferarten (Coleoptera), insbesondere gegen Chrysomeliden, wie z. B. den Blauen Erlenblattkäfer (Agelastica alni) sowie den Kartoffelkäfer (Leptinotarsa decemlineata) besitzt. Es handelt sich als um einen neuen Pathotyp, der als Pathotyp C bezeichnet wird.The invention relates to a new variety of Bacillus thuringiensis, which is referred to as Bacillus thuringiensis var. Tenebrionis (Btt). It could be shown that this strain has an insecticidal action, namely on beetles (Coleoptera), in particular against chrysomelids, such as. B. has the blue alder beetle (Agelastica alni) and the Colorado beetle (Leptinotarsa decemlineata). It is a new pathotype, which is called pathotype C.
Ein weiterer Gegenstand der Erfindung ist ein bakterielles Insektizid, das als aktiven Wirkstoff Bacillus thuringiensis von Pathotyp C, insbesondere, Bacillus thuringiensis var. tenebrionis, oder ein hieraus erhältliches, insektizid wirkendes Präparat bzw. Toxin enthält.Another object of the invention is a bacterial insecticide which contains as active ingredient Bacillus thuringiensis of pathotype C, in particular Bacillus thuringiensis var. Tenebrionis, or an insecticidal preparation or toxin obtainable therefrom.
Die neue Varietät Btt wurde isoliert aus kranken Entwicklungsstadien des Gemeinen Mehlkäfers (Tenebrio molitor L., Coleoptera: Tenebrionidae). Die Haltung der Reinkultur erfolgt bei 25-30oC auf Nähragar in Schrägröhrchen. Trockene Sporen wurden zur Typ-Konservierung eingefroren. Die Determination erfolgte aufgrund morphologischer und biochemischer Merkmale.The new variety Btt was isolated from the developmental stages of the common flour beetle (Tenebrio molitor L., Coleoptera: Tenebrionidae). The pure culture is kept at 25-30 o C on nutrient agar in slant tubes. Dry spores were frozen for type preservation. The determination was based on morphological and biochemical characteristics.
Das isolierte Bakterium produziert auf Nähragar große rauhe, weißliche Kolonien, deren Zellen nach 18 bis 24 Stunden sporulieren. Die vegetative Zelle ist begeißelt, stäbchenförmig und mißt etwa 1,0 x 4 - 8 µm. Sie reagiert grampositiv. Die Sporenmutterzelle enthält neben der subterminal gelegenen ellipsoidalen Spore (0,8 x 1,4 - 1,6 µm) einen parasporalen Kristall von flacher, plättchenartiger Form und mit dem Umriß eines Parallelogramms, Rhombus oder Quadrats (ca. 0,8 - 1,5 µm Kantenlänge).The isolated bacterium produces large, rough, whitish colonies on nutrient agar, the cells of which sporulate after 18 to 24 hours. The vegetative cell is flagellate, rod-shaped and measures approximately 1.0 x 4 - 8 µm. She reacts gram positive. In addition to the subterminally located ellipsoidal spore (0.8 x 1.4 - 1.6 µm), the spore mother cell contains a parasporal crystal of flat, plate-like shape and with the outline of a parallelogram, rhombus or square (approx. 0.8 - 1, 5 µm edge length).
In der vegetativen Phase erfolgt das Wachstum des Bacillus fakultativ auch anaerob. Die Sporulation ist dagegen strikt aerob. Das Temperaturoptimum für das Wachstum der vegetativen Zellen liegt bei 25-30°C. Autoklavieren 15 Minuten bei 1210C (entsprechend 1 bar Überdruck) inaktiviert die Sporen und auch das Kristalltoxin vollständig. Durch UV-Bestrahlung bei 254 nm werden die Sporen bei entsprechender Dosis inaktiviert, nicht aber das Kristalltoxin.In the vegetative phase, Bacillus growth is optionally anaerobic. In contrast, sporulation is strictly aerobic. The optimum temperature for the growth of vegetative cells is 25-30 ° C. Autoclaving for 15 minutes at 121 ° C. (corresponding to 1 bar overpressure) completely inactivates the spores and also the crystal toxin. UV radiation at 254 nm inactivates the spores at the appropriate dose, but not the crystal toxin.
Die biochemischen Leistungen der vegetativen Zellen sind folgende. Aus abbaufähigen Kohlenhydraten (wie z. B. Glucose) wird Gärungssäure und Acetylmethylcarbinol gebildet, aber kein Gas. Außer Glucose werden unter Säuerung Mannose und Saccharose abgebaut, dagegen nicht Lactose, Cellobiose, Salicin, Arabinose, Xylose oder Mannit. Aesculin wird hydrolisiert, desgleichen Stärke. Es können folgende Enzyme nachgewiesen werden: Katalase, Nitratreduktase und Proteinase. Es konnten nicht nachgewiesen werden: Lysin-Decarboxylase, Phenylalanin-Desaminase, Urease und Lecithinase.The biochemical performances of the vegetative cells are as follows. Degradable carbohydrates (such as glucose) produce fermentation acid and acetylmethyl carbinol, but no gas. In addition to glucose, mannose and sucrose are broken down under acidification, but not lactose, cellobiose, salicin, arabinose, xylose or mannitol. Aesculin is hydrolyzed, as is starch. The following enzymes can be detected: catalase, nitrate reductase and proteinase. The following could not be detected: lysine decarboxylase, phenylalanine deaminase, urease and lecithinase.
Für Produktionszwecke im Labormaßstab wurde ein Brutschüttler eingesetzt. In Erlenmeyerkolben von 250 ml wurden 50 ml Medium fermentiert. Das Medium hatte folgende Zusammensetzung: Hefeextrakt 0,5 %, Trypton 0,5 %, Glucose 0 , 1 % , KH2P04 0,08 % in Leitungswasser pH 7,0. Als Bebrütungstemperatur wurde 25 - 30°C gewählt. Im Anschluß an die Sporulation der Kultur, die mikroskopisch kontrolliert werden kann, wurde die Biomasse abzentrifugiert und mit Wasser oder Puffer ausgewaschen. Die Wirkung von Btt auf Coleopteren-Larven folgt dem Schema, das bereits vom Pathotyp A bekannt ist: Es tritt Fraßstopp innerhalb weniger Stunden und Mortalität nach einigen Tagen in Abhängigkeit von der Dosis ein. Die toxische Wirkung beruht darauf, daß das Epithel des Mitteldarmes durch das im Darmsaft gelöste Kristalltoxin nachhaltig geschädigt wird. Die geschädigten Darmzellen lösen sich voneinander und von der Basalmembran und driften in das Darmlumen. Infolge des Zusammenbruchs der Darmschranke können Bakterienzellen in das Hämocoel eindringen und dort den Prozeß der Desintegration durch eine tödliche Septikämie vollenden.A brooder was used for production purposes on a laboratory scale. 50 ml of medium were fermented in 250 ml Erlenmeyer flasks. The medium had the following composition: yeast extract, tryptone 0.5%, glucose 0, 1%, KH 2 P0 4 0.08% in tap water 0.5% p H 7.0. 25-30 ° C was selected as the incubation temperature. Following the sporulation of the culture, which can be checked microscopically, the biomass was centrifuged off and washed out with water or buffer. The effect of Btt on coleopteran larvae follows the scheme already known from pathotype A: feeding stops within a few hours and mortality occurs after a few days depending on the dose. The toxic effect is due to the fact that the epithelium of the middle intestine is permanently damaged by the crystal toxin dissolved in the intestinal juice. The damaged intestinal cells detach from one another and from the basement membrane and drift into the intestinal lumen. As a result of the breakdown of the intestinal barrier, bacterial cells can penetrate the hemocoel and there complete the process of disintegration through fatal septicemia.
Die neue Varietät Btt, Pathotyp C, deren Prototyp aus einer toten Mehlkäferpuppe isoliert werden konnte, wurde bei der Deutschen Sammlung für Mikroorganismen unter der Nr. DSM 2803 hinterlegt. Nach der serologischen Untersuchung gehört die var. tenebrionis, Stamm DSM 2803, zum Serotyp H 8a, 8b.The new variety Btt, pathotype C, whose prototype could be isolated from a dead flour beetle pupa, was deposited with the German Collection for Microorganisms under the number DSM 2803. After the serological examination, the var. Tenebrionis, strain DSM 2803, belongs to the serotype H 8a, 8b.
Die neue Varietät Btt, Pathotyp C, zeigt eine selektive insektizide Wirkung auf Coleoptera, beispielsweise auf den Blauen Erlenblattkäfer oder den Kartoffelkäfer. Die Wirkung ist bei Larven wesentlich stärker als bei Imagines. Keine Wirkung konnte beobachtet werden gegen Lepidoptera, beispielsweise gegenüber der Mehlmotte (Ephestia Kühniella Zell.), Kohlmotte (Plutella xylostella L.) und auch nicht gegenüber Diptera, beispielsweise der Gelbfiebermücke (Aedes aegypti L.). In der folgenden Tabelle sind die Ergebnisse der durchgeführten Biotests zusammengestellt.The new variety Btt, pathotype C, shows a selective insecticidal effect on Coleoptera, for example on the blue alder beetle or the Colorado beetle. The effect is much stronger for larvae than for adults. No effect was observed against Lepidoptera, for example against the meal moth (Ephestia Kühniella Zell.), Cabbage moth (Plutella xylostella L.) and also not against Diptera, for example the yellow fever mosquito (Aedes aegypti L.). The results of the bioassays carried out are summarized in the following table.
Toxische Wirkung von Btt auf verschiedene Coleoptera, Lepidoptera bzw. Diptera
Das eigentliche insektizide Wirkprinzip bei Bacillus thuringiensis ist das Kristalltoxin. Die Kristallmasse kann isoliert werden, indem man bei den sporulierten Kulturen nach der Lyse der Sporangien die Kristalle von den Zellresten und den Sporen trennt und reinigt, beispielsweise über Dichtegradienten.The actual insecticidal principle of action in Bacillus thuringiensis is crystal toxin. The crystal mass can be isolated by separating and cleaning the crystals from the cell residues and the spores in the sporulated cultures after lysis of the sporangia, for example using density gradients.
Bei der Molekulargewichtsbestimmung mit Hilfe der SDS-Polyacrylamidgelelektrophorese nach der Methode von Laemmli [Nature 227, 680 (1970)] werden zwei Hauptbanden bei ca. 65 000 D und ca. 70 000 D gefunden. Kleinere Banden von geringerer Intensität liegen im Bereich von 20.000 bis 40.000 D.When determining the molecular weight using SDS polyacrylamide gel electrophoresis using the method of Laemmli [Nature 227, 680 (1970)], two main bands are found at approximately 65,000 D and approximately 70,000 D. Smaller bands of lower intensity are in the range of 20,000 to 40,000 D.
Mit Hilfe des erfindungsgemäßen Toxins können Antikörper gewonnen werden, die im Immunodiffusionstest nach Ouchterlony keine Kreuzreaktion mit anderen Toxinen des Pathotyps A oder B zeigen.With the help of the toxin according to the invention, antibodies can be obtained which show no cross-reaction with other toxins of the pathotype A or B in the immunodiffusion test according to Ouchterlony.
Das Toxin wird von einem Gen codiert, das in einem Plasmid enthalten ist. Das Toxin wird auch in asporogenen Mutanten produziert, die durch Behandlung mit Ethylmethan-sulfonat erzeugt werden. Die Fähigkeit zur Toxinproduktion kann durch Plasmid- oder Gentransfer auf andere Bacillus thuringiensis Varietäten oder andere Bakteriensysteme übertragen werden. Die insecticide Wirkung beruht auf dem Vorhandensein des plasmidcodierten Toxins.The toxin is encoded by a gene contained in a plasmid. The toxin is also produced in asporogenic mutants, which are generated by treatment with ethyl methane sulfonate. The ability to produce toxins can be transferred to other Bacillus thuringiensis varieties or other bacterial systems by plasmid or gene transfer. The insecticidal effect is based on the presence of the plasmid-encoded toxin.
Zur Anwendung als Insektizid wird Bacillus thuringiensis var. tenebrionis oder ein hieraus erhältliches, insektizid wirkendes Präparat bzw. Toxin in an sich bekannter Weise mit üblichen Additiven (Trägerstoffe, Haftmittel, Netzmittel etc.) versetzt und in eine geeignete Anwendungsform übergeführt. Das so formulierte Insektizid kann in Form eines Spritzpulvers, einer Suspension, als Granulat o. ä. eingesetzt werden. Bevorzugt wird eine gereinigte, sporulierte Kultur in Form einer Suspension unter Zusatz eines Netzmittels, beispielsweise 0,1 % CitowetR (BASF), eingesetzt. Pro Hektar zu behandelndes Feld werden 10 bis 1015, vorzugsweise 1013 bis 1014 Sporen und eine äquivalente Anzahl von toxischen Kristallen versprüht. Auf diese Weise gelingt es zum Beispiel ein Kartoffelfeld 2 bis 4 Wochen lang vor dem Kartoffelkäfer zu schützen. Zum Schutze während der gesamten Wachstumsphase sind weitere Behandlungen in dem genannten zeitlichen Abstand erforderlich. Eine Wirkung gegen Nicht- Zielorganismen, wie beispielsweise gegen Entomophagen und Honigbienen, war nicht zu beobachten.For use as an insecticide, Bacillus thuringiensis var. Tenebrionis or an insecticidal preparation or toxin obtainable therefrom is mixed with conventional additives (carriers, adhesives, wetting agents, etc.) in a manner known per se and converted into a suitable application form. The insecticide formulated in this way can be used in the form of a wettable powder, a suspension, as granules or the like. A purified, sporulated culture is preferably used in the form of a suspension with the addition of a wetting agent, for example 0.1% Citowet R (BASF). 10 to 10 15 , preferably 10 13 to 10 14 spores and an equivalent number of toxic crystals are sprayed per hectare to be treated. In this way, for example, a potato field can be protected from the Colorado beetle for 2 to 4 weeks. For protection during the entire growth phase, further treatments are required at the intervals mentioned. An effect against non-target organisms, such as, for example, against entomophages and honeybees, was not observed.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT84115535T ATE60980T1 (en) | 1983-12-21 | 1984-12-15 | NEW VARIETY OF BACILLUS THURINGIENSIS EFFECTIVE AGAINST COLEOPTERA AND AN INSECTICIDAL PREPARATION AVAILABLE THEREOF. TOXIN AND ITS USE TO COMBAT COLEOPTERA. |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19833346138 DE3346138A1 (en) | 1983-12-21 | 1983-12-21 | BACILLUS THURINGIENSIS VAR. TENEBRIONIS AND AN INSECTICIDALLY EFFECTIVE PRESERVATIVE OR AVAILABLE. TOXIN AND THEIR USE FOR COMBATING COLEOPTERA |
| DE3346138 | 1983-12-21 |
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| Publication Number | Publication Date |
|---|---|
| EP0149162A2 true EP0149162A2 (en) | 1985-07-24 |
| EP0149162A3 EP0149162A3 (en) | 1986-12-30 |
| EP0149162B1 EP0149162B1 (en) | 1991-02-27 |
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| Application Number | Title | Priority Date | Filing Date |
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| EP84115535A Expired - Lifetime EP0149162B1 (en) | 1983-12-21 | 1984-12-15 | Variety of bacillus thuringiensis effective against coleoptera and an insecticidally active preparation obtainable therefrom, or a toxine, and their use to combate coleoptera |
Country Status (11)
| Country | Link |
|---|---|
| US (3) | US4766203A (en) |
| EP (1) | EP0149162B1 (en) |
| JP (2) | JPH0634708B2 (en) |
| AT (1) | ATE60980T1 (en) |
| CA (1) | CA1268133A (en) |
| DE (2) | DE3346138A1 (en) |
| ES (1) | ES8605032A1 (en) |
| HU (1) | HU195724B (en) |
| IL (1) | IL73838A (en) |
| PL (2) | PL154613B1 (en) |
| SU (1) | SU1448995A3 (en) |
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| US4853331A (en) * | 1985-08-16 | 1989-08-01 | Mycogen Corporation | Cloning and expression of Bacillus thuringiensis toxin gene toxic to beetles of the order Coleoptera |
| US5024837A (en) * | 1987-05-06 | 1991-06-18 | Donovan William P | Coleopteran active microorganisms, related insecticide compositions and methods for their production and use |
-
1983
- 1983-12-21 DE DE19833346138 patent/DE3346138A1/en not_active Ceased
-
1984
- 1984-12-15 DE DE8484115535T patent/DE3484196D1/en not_active Expired - Lifetime
- 1984-12-15 AT AT84115535T patent/ATE60980T1/en active
- 1984-12-15 EP EP84115535A patent/EP0149162B1/en not_active Expired - Lifetime
- 1984-12-16 IL IL73838A patent/IL73838A/en not_active IP Right Cessation
- 1984-12-18 PL PL1984270337A patent/PL154613B1/en unknown
- 1984-12-18 PL PL1984251010A patent/PL145191B1/en unknown
- 1984-12-19 SU SU3875505A patent/SU1448995A3/en active
- 1984-12-19 CA CA000470558A patent/CA1268133A/en not_active Expired - Lifetime
- 1984-12-20 HU HU844761A patent/HU195724B/en not_active IP Right Cessation
- 1984-12-21 ES ES538984A patent/ES8605032A1/en not_active Expired
- 1984-12-21 JP JP59268685A patent/JPH0634708B2/en not_active Expired - Lifetime
-
1987
- 1987-03-13 US US07/027,991 patent/US4766203A/en not_active Expired - Lifetime
-
1988
- 1988-05-18 US US07/195,511 patent/US4851340A/en not_active Expired - Lifetime
- 1988-10-18 US US07/259,161 patent/US4973676A/en not_active Expired - Lifetime
-
1993
- 1993-10-13 JP JP5255833A patent/JPH0729895B2/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU548625A1 (en) * | 1976-01-09 | 1977-02-28 | Мордовский государственный университет им.Н.П.Огарева | Strain you option wormwood leaf beetle |
| EP0063949A2 (en) * | 1981-04-27 | 1982-11-03 | Washington Research Foundation | Plasmids coding for B. Thuringiensis crystal protein, their production and their use in creating genetically engineered bacterial strains, the aforesaid strains per se and their use in producing said protein, said protein so-produced, insecticidal formulations comprising the same and a method of producing an insecticidal effect |
| EP0093062A1 (en) * | 1982-04-26 | 1983-11-02 | Institut Pasteur | DNA containing a sequence encoding for a crystal protein or a polypeptide having insecticidal properties, microorganisms transformed with such DNA, compositions containing said crystal proteins, polypeptide or microorganisms |
Non-Patent Citations (4)
| Title |
|---|
| BIOLOGICAL ABSTRACTS, Band 77, Nr. 8, 1984, Seite 6409, Biosciences Information Service, Philadelphia, US; Referenz Nr. 58279; G. CANTWELL et al.: "Activity of the beta-exotoxin of Bacillus thuringiensis var. thuringiensis against the Colorado potato beetle ÄLeptinotarsa decemlineataÜ (Coleoptera: Chrysomelidae) and bacterial mutagenic response as determined by the Ames Test"; & ENVIRON ENTOMOL, 12(5): Pages 1424-1427, 1983 * |
| SOVIET INVENTIONS ILLUSTRATED, Section CH: Chemical, Woche A05, 14. März 1978, Referenz Nr. 9781, Derwent Publications, London, GB and SU-A-548 625 (MORDOV OGAREV UNIV) 09-01-1976 * |
| SOVIET INVENTIONS ILLUSTRATED, Section CH: Chemical, Woche A05, 14. März 1978, Referenz Nr. 9781, Derwent Publications, London, GB; & SU-A1-548 625 (MORDOV OGAREV UNIV) (28-02-1977) * |
| ZEITSCHRIFT F]R ANGEWANDTE ENTOMOLOGIE, Band 96, 1983, Seiten 500-508, Hamburg, DE; A. KRIEG et al.: "Bacillus thuringiensis var. tenebrionis: ein neuer, gegen}ber Larven von Coleopteren wirksamer Pathotyp". * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0213818B1 (en) * | 1985-08-16 | 1991-02-06 | Novo Nordisk A/S | Cloning and expression of bacillus thuringiensis toxin gene toxic to beetles of the order coleoptera |
| EP0221024A2 (en) * | 1985-10-30 | 1987-05-06 | Sandoz Ag | Novel microorganisms |
| EP0221024A3 (en) * | 1985-10-30 | 1988-06-01 | Sandoz Ag | Novel microorganisms |
| EP0238311A2 (en) * | 1986-03-21 | 1987-09-23 | Mycogen Corporation | The use of bacillus thuringiensis to control pests |
| EP0238311A3 (en) * | 1986-03-21 | 1989-07-12 | Mycogen Corporation | The use of bacillus thuringiensis to control pests |
| EP0731170A1 (en) * | 1987-04-29 | 1996-09-11 | Monsanto Company | Insect-resistant plants |
| US6284949B1 (en) | 1987-04-29 | 2001-09-04 | Monsanto Company | Insect-resistant plants comprising a Bacillus thuringiensis gene |
| US6953835B2 (en) | 1987-04-29 | 2005-10-11 | Monsanto Technology Llc | Coleopteran toxin proteins of bacillus thuringiensis |
| WO1991014369A1 (en) * | 1990-03-22 | 1991-10-03 | Novo Nordisk A/S | Use of a coleopteran active bacillus thuringiensis for controlling the cereal leaf beetle oulema melanopa l. (lema melanopa (l.)) |
Also Published As
| Publication number | Publication date |
|---|---|
| ES8605032A1 (en) | 1986-03-16 |
| IL73838A (en) | 1988-11-30 |
| PL145191B1 (en) | 1988-08-31 |
| DE3346138A1 (en) | 1985-07-11 |
| ES538984A0 (en) | 1986-03-16 |
| US4973676A (en) | 1990-11-27 |
| ATE60980T1 (en) | 1991-03-15 |
| CA1268133A (en) | 1990-04-24 |
| US4766203A (en) | 1988-08-23 |
| JPH0729895B2 (en) | 1995-04-05 |
| PL251010A1 (en) | 1985-08-27 |
| EP0149162B1 (en) | 1991-02-27 |
| HUT36690A (en) | 1985-10-28 |
| US4851340A (en) | 1989-07-25 |
| JPH06192027A (en) | 1994-07-12 |
| PL154613B1 (en) | 1991-08-30 |
| HU195724B (en) | 1988-07-28 |
| DE3484196D1 (en) | 1991-04-04 |
| SU1448995A3 (en) | 1988-12-30 |
| JPH0634708B2 (en) | 1994-05-11 |
| EP0149162A3 (en) | 1986-12-30 |
| IL73838A0 (en) | 1985-03-31 |
| JPS60156382A (en) | 1985-08-16 |
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